Lipid mediated plant immunity in susceptible and tolerant soybean cultivars in response to Phytophthora sojae colonization and infection

Background Soybean is one of the most cultivated crops globally and a staple food for much of the world's population. The annual global crop losses due to infection by Phytophthora sojae is currently estimated at $20B USD, yet we have limited understanding of the role of lipid mediators in the adaptative strategies used by the host plant to limit infection. Since root is the initial site of this infection, we examined the infection process in soybean root infected with Phytophthora sojae using scanning electron microscopy to observe the changes in root morphology and a multi-modal lipidomics approach to investigate how soybean cultivars remodel their lipid mediators to successfully limit infection by Phytophthora sojae. Results The results reveal the presence of elevated biogenic crystals and more severe damaged cells in the root morphology of the infected susceptible cultivar compared to the infected tolerant cultivars. Furthermore, induced accumulation of stigmasterol was observed in the susceptible cultivar whereas, induced accumulation of phospholipids and glycerolipids occurred in tolerant cultivar. Conclusion The altered lipidome reported in this study suggest diacylglycerol and phosphatidic acid mediated lipid signalling impacting phytosterol anabolism appears to be a strategy used by tolerant soybean cultivars to successfully limit infection and colonization by Phytophthora sojae. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-04808-z.


Background
The global population is anticipated to increase to almost 9.7 billion by 2050, which will require a 70% increase in food production [1].Food insecurity remains prevalent in many nations despite efforts to improve the production, the quality, and the availability of global food supplies [2].Food insecurity is a major challenge that must be addressed to meet the demands of an ever-increasing global population [3].To fulfill global food and feed requirements, innovative agricultural practices must be developed to enhance food production, availability and accessibility, which in turn will require advanced knowledge in plant pathology from seedling to crop harvest [4].For instance, plant diseases are caused by infectious pathogens such as fungi, viruses, bacteria, and nematodes [4].These diseases lead to significant annual economic losses in maize, potato, wheat, rice, and soybean worldwide accounting for a 40% yield reduction [4,5].Globally, approximately $20B USD are lost annually due to soybean root and stem rot disease caused by the oomycete Phytophthora sojae [6][7][8][9].Soybean root and stem rot are the most devastating threat to seedling and plant survival affecting productivity, particularly during wet growing seasons [6,8].During the susceptible crop growth stages, pathogens can alter an otherwise favourable environment for the plant into unfavourable conditions, leading to significant yield losses [10].The repeated applications and heavy dependence on synthetic chemicals such as fungicides limit effective long-term control of this disease, as well as pose serious environmental and human health risks [11].Reducing the frequency and volume of chemical applications in agricultural crops is one of the primary objectives of plant pathological research.Hence, there is a need to develop innovative disease control systems improving the plant's natural defense mechanisms to build enduring and wide-spectrum disease resistance in crops to improve sustainable agriculture and food security [12,13].
Plants respond to different biotic and abiotic stress conditions through various defense mechanisms that may be either constitutive or induced [4,14].The constitutive system utilizes pre-formed inhibitory chemicals such as alkaloids, saponins, and glycosides, and barriers like wax cuticles, cellulose and suberin to reduce pathogen entry [4,8,15].Induced defense mechanisms are triggered by pathogen ingress causing plants to synthesize compounds or enzymes as a result of pathogen detection.This may occur at the site of infection by processes like the oxidative burst or the hypersensitive response, or the production of chitinases, nitric oxide or phytoalexins [4].The plant cell wall is known to play multiple physiological roles during plant-pathogen interactions.The cell wall structurally consists of non-polysaccharides such as wax, lignin and carbohydrate-based polymers such as hemicellulose, cellulose in form of biogenic crystals, pectin, and chitin [8,16].Both hemicellulose and pectin are mixtures of highly branched polysaccharides in the cell wall of plants that spatially interact with each other [17].The cellulose crystalline are bounded by hemicellulose and lignin [17].These crystalline are crosslinked into a tough network of fibrous molecules that are responsible for mechanical and structural strength of the cell walls and play active role during plant response to environmental stimuli [18,19].Biogenic and biological crystals such as calcium oxalate are biosynthesized in various plant tissues, particularly during physiological and pathological processes [20,21].These crystals serve as support, protection, or defense in plants [20,21].In addition, plant cell walls contain different glycoproteins that are integrated into the matrix that are likely to provide more structural support to cell walls [22].Plant glycoproteins are also involved in some biological functions during plant development and plant innate immunity.Furthermore, the response can be systemic in nature, producing pathogenesis-related proteins or the induction of systemic acquired resistance [23,24].Plants can also adapt to environmental stresses by regulating biochemical, physiological, and molecular properties of their cellular membrane [4,25].Several studies have demonstrated the roles of lipids in plant pathology as part of a complex internal defense mechanism in the fight against infections caused by various pathogens [4,[26][27][28][29].Lipid remodeling is a defence mechanism adopted by plants to counteract pathogen attack [30].Depending on the composition, lipid molecular species can regulate membrane fluidity, permeability, stability, and integrity during a plant's response to pathogenic microorganisms.For instance, free fatty acids (FA) such as linoleic acid and oleic acid, play active roles during biosynthesis of the plant cuticular wax, forming the first barrier against pathogens [27].Lipid metabolites can also function as intracellular and extracellular signal mediators [27,30].Plant lipids include glycerophospholipids (GPL), phytosterols (PST), sphingolipids (SGL), glycoglycerolipids (GGL) and glycerolipids (GL) [4,[31][32][33], and their metabolites are actively involved in plant defence responses against pathogen colonization [34,35].They play important roles in the formation of the membrane interface between plant and the microbial pathogen [34,35].
The GPLs of plant membranes possess two FAs as hydrophobic tails at the sn1 and sn2 carbons and a hydrophilic head group esterified to a phosphate group at the sn3 position of the glycerol moiety.The classes of GPLs include phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS).During plant-pathogen interactions, phospholipid-derived molecules rapidly accumulate and participate in plant signaling and membrane trafficking; they can also activate plant immunity [36,37].For instance, PA acts as a novel secondary messenger in plants and its biosynthesis has been reported to be triggered in response to pathogen attack [39,40].
Plant sphingolipids are structural components of eukaryotic cellular membranes and play essential roles in maintaining membrane integrity [41].They have been recently demonstrated to act as signaling molecules playing crucial functions in the regulation of pathophysiological processes [42][43][44].Studies have demonstrated that sphingolipids play important roles during biotic stress in plants by activating defences against bacterial and fungal pathogens.For instance, the fungus Alternaria alternata f. sp.lycopersici has been shown to activate cell death through disruption of sphingolipid metabolism [45].
Phytosterols are integral components of cellular membranes and the most abundant sterols in plants include campesterol, sitosterol and stigmasterol [46].Phytosterols are actively involved in regulation of membrane fluidity and integrity, and they influence membrane structural properties and physiological functions of plants.For instance, stigmasterol and beta-sitosterol play a vital role during structural formation and mediate cell membrane functions [47].They have also been demonstrated to play essential roles in plant innate immunity against pathogen attack [48].
Galactolipids, including mono-/di-galactosyldiacylglycerol (MGDG and DGDG) are important membrane components in the chloroplasts of eukaryotic plants [49].They play active roles in cell communication, signal transduction, and response to pathogen invasion [35].
Glycerolipids are actively required during cell growth and cell division [50], serve as energy storage for survival, participate in stress responses, and play an important role in reducing pathogenicity [51].During environmental stresses in plants, triacylglycerol (TG) levels increase as a function of the sequestration of toxic lipid intermediates [52].Studies have suggested that diacylglycerols (DGs) serve as signaling molecules during plant growth and development, and in response to stimuli during certain environmental stresses [53,54].In addition, DG and DG kinase are known to activate immunity during plant defence responses to pathogen attack [39].Although the literature is replete with examples of the plant lipidome mediating plant defence, very little is known concerning how plant lipid mediators contributes to either successful colonization or tolerance in the soybean-P.sojae pathosystem.
In addition, lipids serve as structural components of cellular membranes, as well as provide carbon storage or energy, induce cell signaling pathways, and regulate stress responses during pathogenic infection [55,56].Lipid metabolism and lipid homeostasis are known to have systemic effects capable of influencing plant growth, survival and development during plant-pathogen interaction [57].Several studies have documented plantmicrobe interactions and the key changes characteristics of different pathosystems applying various "omics" techniques [29,33,58].However, many questions regarding the communication and biochemical exchange between the plant and microbes during compatible and incompatible interactions is still unclear.Nonetheless, it is well known that pathogenic plant microbial recognition occurs at the cellular membrane.The cell membrane acts as the interface either allowing advantageous resource exchange or inhibiting interaction, as well as cell-cell communication through downstream signaling cascades [59,60] mediated by anabolic or catabolic lipid metabolic reactions.Furthermore, the cell wall is the outermost boundary of the plant cell that either act as a preform or induced barrier to limit colonization upon microbe recognition and penetration [61] as the first line of defense.The cell membrane acts as a major biosensor critical in the perception and signaling responses to initial microbe recognition and downstream responses.The lipids in the cell membrane can be remodeled on the order of seconds to milliseconds or hours as part of the cascade of reactions mounted by the host plant to limit infection and colonization [62].Concomitant with the altered membrane lipid metabolism, there are physical barriers on the plant cell surfaces that are either reinforced or induced to further limit the interaction between the host and microbe.These physical structures include calcium deposits, biogenic crystals, reinforced cell walls etc. that can also act as signaling molecules or sources of elicitors for recognition, thereby activating defense response against microbial colonization [29,63,64]".How these cell morphological changes converge as part of a lipid mediated plant immune response is a matter of great interest in many plant pathogen interactions or pathosystem, including the soybean -P.sojae pathosystem.We hypothesized that the morphological properties of the tolerant cultivar would suggest a mechanism for disease tolerance when compared to the morphological response in the susceptible cultivar and that the lipid mediated plant immunity in a P. sojae-tolerant soybean cultivar would fluctuate more than those of a P. sojae-susceptible cultivar following pathogen infection.To this end, we assessed the histochemical properties and lipidome of soybean root and stem to understand the functions of lipid mediators in the response of susceptible and tolerant soybean cultivars to pathogen colonization and infection.

Histological changes in the root of both soybean cultivars infected with Phytophthora sojae.
To understand the morphological structure of soybean root and how changes occur in the morphology of the root during interaction with pathogens governing tolerance, scanning electron microscopy (SEM) was used to provide detailed images of soybean root morphology.Scanning electron microscopic images demonstrated opening in the cortex of the roots (Fig. 1a-c), the intact epidermis and cortical cell (Fig. 1d, e), degradation and crack in the cortical cells during interaction between soybean and P. sojae (Fig. 1f, g), and closing-up of degradation, crack in the inner cortical side of epidermis and cortex (Fig. 1h, i) and the relative abundance of soybean root segments (Fig. 1j).The tiny openings in the cortical cells were revealed in the non-infected and infected of both susceptible and tolerant soybean cultivars.Meanwhile, there were more opening in the infected susceptible cultivar than in the infected tolerant cultivar (Fig. 2a-d) similar changes in relative abundance of the tiny openings were also present in the cortical cells of the soybean root (Fig. 2a and e).
The SEM also revealed alterations of xylem walls.The cell walls of vascular cylinder of the susceptible cultivar were observed to be degraded after infection compared to the tolerant cultivar (Fig. 3a-d).It was seen that the vascular cylinder of tolerant cultivar was made up of thick cell walls demonstrating a physical response to confine or halt the pathogens from spreading further into the vascular cylinder compared to that of susceptible cultivar following infection with P. sojae (Fig. 3b, d) and the relative abundance of cells with thickened cell walls in the vascular cylinder of soybean root (Fig. 3e).
The SEM micrographs show biogenic crystals of different morphologies in various locations in the soybean roots of both susceptible and tolerant cultivars.Biogenic crystals of various morphologies are present in the xylem in copious amounts in the non-infected and infected susceptible cultivar and these crystals varied greatly in sizes (Fig. 4a, b).However, the amounts of the biogenic crystals observed in non-infected susceptible root remained unchanged in the infected susceptible root.(Fig. 4a, b).In contrast, the crystals were observed to be present in copious amounts in the root of non-infected tolerant cultivar relative to the root of infected tolerant cultivar (Fig. 4c, d).Therefore, it was observed that biogenic crystals found in the roots of the infected susceptible cultivar were present in more copious amounts than in the infected tolerant cultivar (Fig. 4b, d) and vise-visa to their non-infected counterparts (Fig. 4a, c), the relative abundance of biogenic crystals in the vascular cylinder of soybean root (Fig. 4e).

Lipid composition of the soybean cultivars in response to P. sojae infection
We applied a multi-modal lipidomics approach using UHPLC-C30RP-HESI-HRAM-MS/MS to obtain a detailed understanding of how susceptible and tolerant soybean cultivars remodeled their lipid mediators to successfully limit infection by P. sojae using 10-day old seedlings as a model.The results confirmed as hypothesized that there are significant alterations in the root and stem lipidomes within and between susceptible and tolerant soybean cultivars following inoculation with pathogenic P. sojae (Tables 1, 2).Representative chromatograms and mass spectrum demonstrating the separation of the membrane and storage lipids present in the root and stem of the soybean cultivars evaluated (negative and positive ion modes) is shown in Fig. 5.The chromatograms of separated membrane lipids in negative ion mode are shown in Fig. 5a.The extracted ion chromatogram of m/z 671.46, 802.56 and 833.52 precursor ions of the three selected polar lipids are shown in Fig. 5b.The MS 2 spectrum of m/z 671.46 identified as PA 16:0/18:2 [M-H] − is depicted in Fig. 5c.
For example, m/z 152 represent the glycerol moiety (head group) in PA and m/z 255 and 279 represent C16:0 and C18:2 fatty acids present in PA 16:0/18:2 (Fig. 5c).The same convention was used in identifying the other lipids present in Fig. 5.This included m/z 802.56 identified as PC 16:0/18:2 [M + HCOO] − in Fig. 4d, m/z 833.52 representing PI 16:0/18:2 [M-H] − in Fig. 5e.Together, these accounted for some of the main membrane lipids identified in the soybean plant tissue.Similarly, a chromatogram demonstrating the separation of GLs from the stem of the soybean cultivar in the positive ion mode is shown in Fig. 5f.The extracted ion chromatogram of m/z 630.51, 890.72 and 892.74 representing the precursor ions of the three selected GLs are depicted in Fig. 5g.The MS 2 spectrum of m/z 630.51 identified as DG 18:3/18:3 [M + NH 4 ] + is depicted in Fig. 5h, the MS 2 spectrum of m/z 802.56 identified as TG 18:3/18:3/18:3 [M + NH 4 ] + is depicted in Fig. 5i, and the MS 2 spectrum of m/z 833.52 representing TG 18:3/18:2/18:3 [M + NH 4 ] + is depicted in Fig. 5j.These species account for some of the major GLs identified in the plant tissue.Further diagnostic chromatograms demonstrating the characteristic product ions of some biomarkers differentiating the root and stem lipidomes of the soybean cultivars, identified Fig. 4 Scanning electron microscopy showing the presence of biogenic crystals in the root tissues of susceptible and tolerant soybean cultivars when inoculated with P. sojae.aCross section of the non-infected susceptible soybean cultivar, (b) Cross section of the infected susceptible soybean cultivar, (c) Cross section of the non-infected tolerant soybean cultivar, (d) Cross section of the infected tolerant soybean cultivar.Yellow arrows denote the presence of biogenic crystals in the vascular cylinder in copious amounts in both non-infected susceptible and tolerant cultivars.Biogenic crystals were present in the vascular cylinder in copious amounts in the non-infected and infected susceptible cultivar, and in non-infected tolerant cultivar but their quantity was reduced in infected tolerant cultivar.Bars: (a-d) 40 µm.Bar charts indicating the relative abundance of biogenic crystals in the vascular cylinder of soybean root as mean ± SE (e; n > 5).Significant differences between treatments showing biogenic crystals in the vascular cylinder of soybean root are indicated using letters (a-d) on top of the bars as described by Fisher's LSD and ANOVA (α = 0.05) from their lipid biochemical network (Table S2, Figs S4, S5, S6, S7 and S8).

Modification of membrane lipids in soybean cultivars in response to P. sojae infection
An analysis of membrane lipids in soybean root and stem tissues following infection with P. sojae was performed to determine changes and modification of membrane lipids during host-pathogen interaction.Figures 6a-d and 7a-d demonstrate the changes that occurred in membrane lipids during host-pathogen interactions.Based upon the membrane lipid molecular species observed, we conducted PLS-DA to determine the most important membrane lipid molecular species with influential loadings (Figs.6a, b and 7a, b) segregating the tolerant from the susceptible cultivar based on pathogen challenge.The model quality (Q 2 ) represents 95% and 96% variability in root and stem, respectively (Fig. 6a, 7a).The result from the PLS-DA observation plot showed the segregation of the susceptible and tolerant soybean cultivars before and after infection into four distinct groups that are in accordance with the root and stem membrane lipid molecular species (Fig. 6b, 7b).The root membrane lipid molecular species (Fig. 6b) separated the treatments into four distinct quadrants (Q).Quadrant 1 contained the lipid molecular species associated with Conrad root control (CRC) treatment, Q-2 contained Conrad root inoculated (CRI) treatment, Q-3 contained OX760-6 root control (ORC) and Q-4 had the OX760-6 root inoculated (ORI) treatment, respectively.Similarly, the changes in soybean stem (Fig. 6b), lipid molecular species separated the treatments into 4 distinct quadrants (Q-1, Q-2, Q-3 and Q-4) consisting of Conrad stem control (CSC), Conrad stem inoculated (CSI), OX760-6 stem control (OSC) and OX760-6 stem inoculated (OSI) treatments, respectively.Based upon Component 3, which demonstrated the highest variation in the data (Figs.6a, 7a), 22 membrane lipid molecular species from root tissue and 21 membrane lipid molecular species from stem tissue were selected for further analysis.Heat maps (Figs.6c, 7c) were generated for the lipids with influential loadings accounting for the genotype and treatment segregation to further classify the treatments based on the altered membrane lipidome following infection.The cut-off value for variables important in projection (VIP) scores was defined as > 1 [32,65].The 22 important root membrane lipid molecular species and 21 important stem membrane lipid molecular species were selected based on VIP scores greater than 1.The output from the heat map analysis showed four different clusters of the soybean root and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated root membrane lipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of root membrane lipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are root membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root membrane lipid species in each of the bar chart (Fig. 5d) beside the heat map; and (d) Bar charts describe the relative abundance of root membrane lipid species as a mean nmol% ± SE (n = 4).Significant differences between root membrane lipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are root membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760- and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated stem membrane lipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of stem membrane lipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are stem membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem membrane lipid species in each of the bar chart (Fig. 4d) beside the heat map; and (d) Bar charts describe the relative abundance of stem membrane lipid species as a mean nmol% ± SE (n = 4).Significant differences between stem membrane lipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are stem membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem membrane lipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected) and stem membrane lipid molecular species following inoculation with P. sojae (Figs.6c, 7c).

Modification of glycerolipids in soybean cultivars in response to P. sojae infection
We also analysed GL in soybean root and stem tissues following infection with P. sojae to determine whether their levels and composition were altered during hostpathogen interaction (Figs.8a-d, 9a-d).Triacylglycerols and DGs were observed to be the major GLs present regardless of soybean cultivar.We next performed PLS-DA to identify the most important TG and DG species with influential loadings (Figs.8a, b, 9a, b) segregating the tolerant and susceptible soybean cultivars in their response to P. sojae colonization and infection.The model quality (Q 2 ) represents 80% and 83% variability in root and stem, respectively (Fig. 8a,  9a).The result from the PLS-DA observation plot showed the segregation of the susceptible and tolerant soybean cultivars that were infected or not infected with the pathogen into four distinct quadrants based on the levels of GL molecular species (Figs.8b, 9b).The root GL molecular species (Fig. 8b) separated the treatments into and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated root glycerolipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of root glycerolipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are root glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root glycerolipid species in each of the bar chart (Fig. 7d) beside the heat map; (d) Bar charts describe the relative abundance of root glycerolipid species as a mean nmol% ± SE (n = 4).Significant differences between root glycerolipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are root glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root glycerolipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected) and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated stem glycerolipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of stem glycerolipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are stem glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem glycerolipid species in each of the bar chart (Fig. 8d) beside the heat map; and (d) Bar charts describe the relative abundance of stem glycerolipid species as a mean nmol% ± SE (n = 4).Significant differences between stem glycerolipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are stem glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem glycerolipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected) four distinct quadrants.Quadrants 1-4 were composed of the GL molecular species of CRC, CRI, ORC and ORI treatments, respectively.Similar to the changes in soybean stem (Fig. 9b), GL species separated the treatments into 4 distinct quadrants (Q1-Q4) consisting of the GLs from CSC, CSI, OSC and OSI, respectively.
Based upon component 3 which explained the highest level of variation in the data (Figs.8a, 9a), 27 GL molecular species from root tissues and 28 GL molecular species from the stem tissue with VIPs greater than 1 were selected for further multivariate analysis.Heat maps (Figs.8c, 9c) were next generated for the lipids with influential loadings accounting for the genotype and treatment segregation to further classify the treatments based on the altered GL in the infected tissue.The output from the heat map analysis showed four different clusters of the soybean root and stem membrane lipid molecular species following inoculation with P. sojae (Figs.8c, 9c).The heat map clustered GL species into two main groups, G1 and G2, and four sub-groups (G1A, G1B, G2A and G2B).These groupings distinguished the GL lipid molecular species in the root of the susceptible cultivar (ORC and ORI) from those of the root of the tolerant cultivar (CRC and CRI), as well as the stemderived GL lipid molecular species from both the susceptible (OSC and OSI) and tolerant cultivar (CSC and CSI) (Figs. 8, 9).

Discussion
Infectious pathogens usually colonize plant host to obtain nutrients for their own survival and frequently infect plant tissue such as leaves, stems, or roots, facilitating likely spread within the entire plant [66,67].In addition, pathogen can particularly colonize cell types like the root epidermis, phloem or cortical cells and spread to vascular cylinder [68].Although pathogen invasion is normally specific to particular organs, and they gained entry at a distant site [66,67].For instance, R. solanacearum entered plant root through root surface or root tips, wounds, and secondary emerging points of roots as penetration sites; it then progressed through the cortex, then spread to the vascular cylinder and finally colonized and infected the entire plant [66,67,69].It seems that, upon contact with the soybean roots, P. sojae is recognized by the tolerant cultivar and activated defence responses against pathogen.It is normally accepted that the activation of defence response by host plant could have a major effect on plant growth and development, although the precise underlying strategies are unknown.Besides, the observation that infection of root by P. sojae involves morphological changes in certain cell types (Figs. 1).This makes it possible that the overall root development could be changed by the P. sojae colonization and infection, either as a result of the activation of anti-fungi responses by tolerant plant or following active control by the pathogen.It was previously demonstrated that pathogens are capable of penetrating intact roots, and move up to the xylem tissue, and they can also penetrate through wounds or natural apertures to older parts of root and hypocotyl tissue [70] (Fig. 2).Plant resistance to pathogen invasion via colonizing the xylem may be correlated to the thickening of the xylem cell walls [71].However, lipids are critically involved in pathophysiological properties during plant-pathogen interactions, and the alterations in lipid compositions and their functions The biochemical lipid network demonstrates fold differences in 21 stem membrane lipid molecular species and 28 glycerolipid molecular species following inoculation with P. sojae.Lipid SMILES identifiers were used to calculate PubChem molecular fingerprints and structural similarities.Mapped networks, displaying significance of fold differences in lipids were calculated for all comparisons.Network visualizations display lipids connected based on structural Tanimoto similarity ≥ 0.8 (edge width: 0.8 to 1.0).Node size displays fold differences of means between comparisons and color shows the direction of change compared to control (orange: increased; blue: decreased; gray: inconclusive).Node shape displays lipid structural type (rounded square: membrane lipids; circle: glycerolipids).Lipids displaying significant differences between treatment groups (p ≤ 0.05) are denoted with black borders can have significant effects on plant morphology and plant's response to environmental stressors [20,21].For instance, lipids enhance the structural integrity of plant cells to involve in cell signaling cascade that activate plant defense responses [20,21].This was implicated in the current study where the cell wall of vascular cylinder of the infected tolerant cultivar was observed to be thicker compared to the cell wall of vascular cylinder of the susceptible cultivar when infected with P. sojae (Fig. 3).Likewise in plants, crystals may be present and distributed within a single or multiple tissues and varies among plant species though there are no generalities about where crystals can be generated in plants [72] (Figs. 4).It has been generally predicted that the morphology and the distribution of crystals are strictly regulated by plant genes and a specific species will generate a particular type of crystal [72] (Figs. 4).For instance, calcium sulfate crystal and calcium oxalate were found in almost all plant tissues including sclerenchyma, parenchyma and mesophyll, and locations of types of crystals may not or may be tissue-specific [73].Also, certain functions have been proposed for crystal formation in plants.For instance, in Acacia species, crystals consisting of magnesium, calcium, barium, and strontium are used to regulate the levels of these micronutrients in metabolic partitions in the plants and to avert toxicity [74].Likewise, biogenic and biological crystals are generally accepted to play some physiological and pathological roles such as support, protection, and defense in plants [20,21].Studies have demonstrated that anatomy and physiological functions of vascular cylinder is critically important to plant defense against pathogens [75].This study demonstrated interesting information about the system of compartmentalization of P. sojae in the root of soybean cultivars and recognizes the anatomy of xylem as a major factor of disease resistance.In the root of tolerant cultivar, compartmentalization may be contributory to wall-off P. sojae and ensure that the physiological functions and integrity of cellular structures are maintained.
As essential components of cellular membranes, lipids are involved in various physiological roles including as structural components of cellular membranes, cell Fig. 12 Lipid structural similarity network displaying differences in root and stem membrane lipids and glycerolipids in susceptible and tolerant soybean cultivars inoculated with P. sojae.a Lipids from inoculated root tissue of susceptible (ORI) versus tolerant (CRI) soybean cultivars inoculated with P. sojae; and (b) Lipids from inoculated stem tissue of susceptible (OSI) versus tolerant (CSI) soybean cultivars inoculated with P. sojae.The biochemical lipid network demonstrates fold changes in 22 root membrane lipid molecular species and 27 glycerolipid molecular species, and 21 stem membrane lipid molecular species and 28 glycerolipid molecular species following inoculation with P. sojae.Lipid SMILES identifiers were used to calculate PubChem molecular fingerprints and structural similarities.Mapped networks, displaying significance of fold differences in lipids were calculated for all comparisons.Network visualizations display lipids connected based on structural Tanimoto similarity ≥ 0.8 (edge width: 0.8 to 1.0).Node size displays fold differences of means between comparisons and color shows the direction of change compared to control (orange: increased; blue: decreased; gray: inconclusive).Node shape displays lipid structural type (rounded square: membrane lipids; circle: glycerolipids).Lipids displaying significant differences between treatment groups (p ≤ 0.05) are denoted with black borders signaling, storage of energy, and membrane trafficking.In plants, alterations in lipid composition have been reported in response to pathogenic stress conditions [76].Biotic stress have been reported to profoundly alter the lipidome in plants [77].Additionally, Ferrer et al. [78] demonstrated that alterations in the relative composition of PSTs in cellular membranes affect their biophysical properties and hence their physiological functions.The results we describe here indicate significant alterations in lipid mediators in both a tolerant and a susceptible soybean cultivar in response to P. sojae infection.Specifically, in the pathogen-treated plants, we observed significantly higher levels of major GPLs and GLs (DGs and TGs) in the tolerant cultivar, whereas StEs and CmEs were found to be higher in quantity in the susceptible cultivar.More interestingly, these classes of lipids varied in a similar manner in the root and stem of each cultivar in response to pathogen infection, which is in line with the literature [77,79,80].For example, similar trends were observed for the lipidome of eggplants (Solanum melongena) resistant to Fusarium wilt infection [79], demonstrating the significant difference in the levels of lipid profiling of the susceptible and tolerant eggplants to Fusarium disease and this ensured the essential roles of the lipids in resistance strategy against infection [79].The increased lipid levels in tolerant cultivars serve as energy stores and provide a buffer to stress; the stored lipids could act as additional energy that keeps the plants from shifting to proteolysis and then cell death [80].
The biosynthesis and lipid composition of cellular membranes play an essential role in the physiological functioning of plants [81].During growth, plants adapt to adverse stress conditions through the remodelling of lipid membranes resulting from alterations in the fatty acid content and, consequently, the biosynthesis of lipids [81].Several studies have demonstrated that high levels of lipid remodeling in plant membrane lipids under different adverse conditions result in resistance to environmental stressors [81].
Our results clearly show that there are differences in both membrane and storage lipid mediators in tolerant and susceptible soybean cultivars in response to P. sojae infection.For instance, we observed higher levels of 18:2 and 18:3 fatty acyl-enriched phospholipid and sterol molecular species in the membrane lipids of the root and stem from the tolerant cultivar when challenged with the pathogen, in contrast to lower C18:2 and C18:3enriched molecular species in tissues from the susceptible cultivar (Figs. 6, 7).Several studies have indicated that during pathogen infection, accumulated phospholipidderived molecular species participate in plant signaling and membrane trafficking, that can induce immunity in plants [36,37].For instance, PA is known as a secondary messenger in plants and its synthesis has been reported to be induced in response to pathogen attack [39,40]".In line with this, the current study has demonstrated significant accumulation of PA molecular species such as PA16:0/18:2, PG16:0/18:2, PI16:0/18:3, PC16:0/18:2, PS16:0/18:1, PS16:0/18:2, PA18:3/18:3 and PG16:0/16:0 (Figs. 6 and 7) in infected root and stem of tolerant soybean cultivar but no significant changes in infected susceptible cultivar [39,40].Similarly, lipid metabolism in guard cells have been attributed to an 18:3 plant species, Vicia faba [82].Guard cell protoplasts from V. faba have been demonstrated to generate eukaryotic lipid molecular species [82].Guard cells are known to contain high level of the triacylglycerols biosynthesized by the eukaryotic lipid metabolic pathway [82].Recently, it has been established that triacylglycerols stored in guard cells are used to generate adenosine triphosphate required for light-induced stomatal opening.
In contrast, the StEs were significantly higher in the root and stem from the susceptible cultivar challenged with P. sojae infection but were significantly lower in the susceptible control plants and in the tolerant cultivar under both treatment conditions (Tables 1, 2; Figs. 6, 7).This is in agreement with a recent study which demonstrated the role of sterols in disease resistance [83].Stigmasterol ester was identified as a factor of susceptibility in Arabidopsis, as inhibition of its biosynthesis resulted in increased resistance to Pseudomonas syringae [83,84].Following inoculation with pathogenic microorganisms, plants generate an array of metabolic changes that potentially contribute to induce resistance or even enhance susceptibility.In this study, we observed that accumulation of stigmasterol is an important plant metabolic process that occurs upon pathogen infection in soybean-P sojae pathosystem.For instance, we noticed the significant accumulation of StE 19:1, AcHexCmE 18:3, CmE20:2, AcHexSiE16:0, AcHexSiE18:1, SiE18:3, AcHexSiE16:2 and AcHexCmE16:0 in infected root and stem of susceptible soybean cultivar but no significant changes in infected tolerant cultivar (Figs. 6 and  7).This is consistent with previous reports showing β-sitosterol and stigmasterol accumulation promoted Arabidopsis susceptibility to P. syringae [84],through the enzyme CYP710A1.Stigmasterol is biosynthesized from β-sitosterol via cytochrome P450 CYP710A1 [84].For instance, Arabidopsis cyp710A1 mutant lines compromised in pathogen-inducible expression of C22 desaturase and associated stigmasterol accumulation are highly resistant to both virulent and avirulent P. syringae strains compared to the wild-type plants, and exogenous utilization of stigmasterol impaired this resistance phenotype.Thus induced sterol desaturation in susceptible soybean cultivar appears to favours pathogen multiplication and plant susceptibility [84].The formation of stigmasterol in leaves is induced by recognition of bacterial pathogenassociated molecular patterns and synthesis of reactive oxygen species, but is independent of the jasmonic acid, salicylic acid or ethylene-associated signalling pathways [84].Through analysis of mutants and application of exogenous sterol, it was demonstrated that an increase in the ratio of stigmasterol to β-sterol in leaves reduces specific defence responses in Arabidopsis, and consequently makes the plants more susceptible to P. syringae [4,48].These were in line with the results obtained in this study, and these modes of action may account for the higher resistance of the tolerant cultivar to pathogen infection.
Pathogenic fungi can secrete various extracellular enzymes that are involved in pathogenicity [85].For example, secreted lipases from fungal pathogens are involved in the penetration of plant barriers such as the wax cuticle.Similarly, fungal lipases are capable of degrading storage and signaling lipids via the release of secondary messengers.The significant decrease in the TG molecular species such as TG 18:1/18:1/18:1, TG 18:0/18:0/18:0, TG 18:0/16:0/18:1, TG 16:0/18:3/18:3, TG 16:0/16:0/18:3, TG 10:0/12:0/14:1 and TG 10:0/10:0/100 (Figs. 8 and 9) in the soybean susceptible cultivar in this study could be a result of increased lipase activity during infection.For instance, lipases hydrolyze carboxyl esters in TGs liberating fatty acids and glycerol [86].This agrees with the fact that lipases appear to function as virulent factors in plant pathogens.More interestingly, the tolerant cultivar demonstrated significantly higher DG levels such as DG15:0/16:0, DG16:0/14:0, DG18:0/16:0, DG18:0/18:0 and DG16:0/18:1 (Figs. 8 and 9) in response to pathogen infection, but there was no observed difference in TG levels.DGs are primarily derived either from TGs through TG lipases or from PAs by phospholipase activity [79,87]".These findings suggest that soybean root lipidome and metabolism is remodeled in response to pathogen colonization and infection and appears to be part of the successful strategy used by the host in this pathosystem to survive pathogen attack.The lipid biochemical network demonstrated significant alterations in lipid mediators in both cultivars in response to P. sojae infection.The head group and FA composition of complex lipids are a useful proxy for localization and biological function [88].Networks display increased density in connectivity between biochemically related groups of lipids and the lipid biosynthesis metabolism pathway in the tolerant soybean cultivar as defense response to pathogen inversion.Generally, there is dearth of information on the role of lipid mediators in determining either incompatible or compatible interactions in the soybean-P sojae pathosystem during host-pathogen interaction.The unique biomarkers between the susceptible and tolerant cultivars including the production of DG molecular species, which was well pronounced in tolerant cultivar than susceptible (Figs. 10, 11 and 12).Studies have demonstrated that signaling enzymes, diacylglycerol kinases (DGKs) play important roles in response to biotic stress by phosphorylating DG to synthesis PA (Fig. 13) and both PA and DG are lipid mediators during physiological process [89].Our findings from this study demonstrate that lipid mediators and signalling possibly involving DG could play a significant role in pathogen resistance in the tolerant soybean cultivar.Also, DG signally related to TG hydrolysis which was differentially demonstrated between susceptible and tolerant soybean cultivars when challenged with pathogen (Figs. 8 and 9).Study has demonstrated that TG is accumulated in plant tissues due to TG turnover, as a result of disruption of SUGAR-DEPENDENT1, a cytosolic lipase accountable for TG hydrolysis in lipid droplets into free FAs and DG and consequently enhance TG accumulation in plant tissues [90].Fan et al. [91] demonstrated that TG accumulation plays important role, thus buffering homeostasis of lipid and protecting plant cells against lipotoxic death as a results FA overload and can be as a remodeling of robust membrane in response to stresses.Phytosterols also known to play important role in plant innate immunity against pathogen attack [48].However, the odd chain FAs that appear in the lipid profile are usually and maybe fungal origin and they are mainly unique to the infected soybean [92].The extracted ion chromatograms of the odd chain FAs are shown in Fig. S1 and MS 2 spectrum of m/z 856.73 identified as TG 15:0/18:2/18:3 [M + NH 4 ] + is depicted in Fig. S2.
Lipid biosynthesis in soybean cultivars follow common routes where FAs are generated from plastid, transported to the endoplasmic reticulum (ED) [38], which starts with the addition of fatty acyl-CoA leading to biosynthesis of lysophosphatidic acid (LPA) and the reaction is catalyzed by glycerol phosphate acyltransferase (GPAT) and is a rate limiting-step for PA biosynthesis.In ED, PA biosynthesis occurs by addition of fatty acyl-CoA to LPA via lysophosphatidic acid acyltransferase (LPAAT) to form central precursor PA by which several GPLs are synthesized (Fig. 13).The first step in GPLs biosynthesis involves the hydrolysis of the phosphate group from PA to generate DG by phosphatidic acid phosphatase (PAP).The resulting DG is later phosphorylated by DGKs to PA, which is subsequently reused in biosynthesis of GPLs.Also, DG acts as a precursor for biosynthesis of primary form of storage energy, TG [93].The isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) generated via cytosolic mevalonate (MVA) pathway are primarily used for the biosynthesis of phytosterols [94,95].Our results demonstrate novel information about pathogen-stress responses in the root and stem of both soybean cultivars, which can be put within the broad context of plant lipid mediators.The metabolic pathway of relative abundance of GPL, PST and GL biosynthesized in the root and stem of the susceptible and tolerant soybean cultivars when These lipid classes could be used as biomarkers for disease resistance or susceptibility by soybean cultivars.Based on our understanding, this is the first report of lipid alteration in soybean root and stem in response to P. sojae infection.

Plant growth and inoculation method
A virulent strain of P. sojae race 2 (strain P6497) obtained from the London Research and Development Center, Agriculture and Agri-Food Canada (AAFC-LRDC; London, ON, Canada) was used as inoculum.The oomycete was cultured and maintained aseptically for 8 days on 26% V8-juice agar (8400 mg agar, 1600 mg CaCO 3 , 156 mL V8-juice [Campbell Soup Company, Toronto, ON, Canada], and 440 mL of distilled water).Seeds of soybean cultivars Conrad (P.sojae-tolerant) and OX760-6 (P.sojae-susceptible) were obtained from AAFC-LRDC (London, ON, Canada).The seeds were surface disinfected for 5 min using 0.5% sodium hypochlorite (Commercial Javex Bleach; Clorox Co., Brampton, Ontario, Canada) and rinsed with distilled water several times.The seeds were then soaked for 12 h in distilled water before seeding.Agar disks containing P. sojae (isolate P6497) were carefully cut from each agar plate using an empty cup as a clamp.Each disk was fitted into the bottom of empty wax-paper cups with a top diameter of 8.5 cm and 15.0 cm deep (Merchants Paper Company, Windsor, ON, Canada) and overlaid with medium-grade vermiculite.Drainage holes were created in the bottom of the cups.The imbibed seeds were planted in the medium-grade vermiculite.Six soybean seedlings from each cultivar were inoculated with P. sojae and another six from each cultivar were mock-inoculated (sterile V8-juice agar disks without any P. sojae culture) in a cup as the control.The plants were then grown and inoculated using agar disk placed in the bottom of the cups for 10 days and seedlings (10 days old) used for subsequent analysis and lab experimentation.Susceptible plants started showing symptom of infection at 6 days after growth in the medium containing the agar disks [7,8].The plant growth experiment was performed in a growth chamber (Biochambers MB, Canada) at Grenfell Campus, Memorial University of Newfoundland, under controlled growth conditions of 16 h light at 25 °C and 8 h dark at 20 °C, and relative humidity of 60%.Seedlings were watered daily 4 days after seeding with one-quarterstrength Knop's solution [8].The whole seedlings were collected 10 days after growth and stored at -80 °C until further analysis.

Preparation of samples for scanning electron microscopy
Soybean roots were collected from both non-inoculated (control) and inoculated plants of susceptible and tolerant soybean cultivars.The samples were rinsed with distilled water before further processing.Free-hand cross sections of the root were cut using a razor blade to a length of approximately 5 mm.Thin sections were mounted to aluminum stubs using colloidal graphite adhesive (Permatex, Canada, Incorporated).The samples were exposed to a temperature of -4.9 °C on a Peltier cooling stage to minimize differences in structure, while in the vacuum chamber.The images of the samples were collected using an environmental scanning electron microscope (ThermoFisher Quattro S with ESEM), to study the morphological properties of roots infected with P. sojae.High-resolution images were collected from 9-10 mm to 5-100 µm (magnification 788-8000X with the pressure 50-428 Pa).

Method of lipid extraction
Soybean seedlings prepared as above were incubated in boiling isopropanol for 10 min.Lipid extraction was conducted by weighing 100 mg each of root and stem from each sample type, and 1 mL MeOH containing 0.01% butylated hydroxytoluene was added to each sample.Four replications of each combination of treatment (inoculated or control), cultivar (susceptible or tolerant), and tissue (root or stem) combination were performed.The tissues were then homogenized using a probe tissue homogenizer until completely dissolved.Following homogenization, 800 µL water and 1000 µL chloroform were added along with PC 14:0/14:0 as internal standard.Each sample was thoroughly vortexed and centrifuged at 3000 rpm for 15 min at room temperature.The organic layers were transferred to pre-weighed 4 mL glass vials with PTFE-lined caps (VWR, Mississauga, Canada).The samples were then dried under a gentle stream of nitrogen and the sample vials reweighed to determine the quantity of recovered lipids.The recovered lipids from each sample were re-suspended in 1000 µL solvent (2:1 v/v chloroform: methanol) and stored at -20 °C until lipid analysis using ultra high-performance liquid chromatography coupled to heated electrospray ionization high resolution accurate mass tandem mass spectrometry (UHPLC-C30RP-HESI-HRAM-MS/MS).

Lipid analysis using UHPLC-C30RP-HESI-HRAM-MS/MS
The method of lipid analysis was as described previously [32,96,97].Lipids extracted from the soybean roots and stems were separated using an Accucore C30 reverse phase (C30RP) column (150 × 2 mm I.D., particle size: 2.6 µm, pore diameter: 150 Å; ThermoFisher Scientific, ON, Canada) applying the following solvent system: Solvent A (40: 60 v/v H 2 O and acetonitrile), and Solvent B (1:10: 90 v/v/v water: acetonitrile: isopropanol).Both solvents A and B consisting of 0.1% formic acid and 10 mM ammonium formate.The conditions for the separation using UHPLC-C30RP were as follows: oven temperature of 30 °C, flow rate of 0.2 mL/min, and 10 µL of the lipid mixture suspended in 1: 2 v/v methanol: chloroform was injected into the instrument.The system gradient used for the separation of lipid classes and molecular species were: 30% solvent B for 3 min; solvent B increased over 5 min to 43%, then increased in 1 min to 50% B and to 90% B over 9 min; and from 90 to 99% B over 8 min; and finally maintained at 99% B for 4 min.The column was re-equilibrated to 70% solvent A for 5 min to re-establish the starting conditions before injection of each new sample.Lipid analyses were performed using a Q-Exactive Orbitrap high-resolution accurate mass tandem mass spectrometer (Thermo-Scientific, Berkeley, CA, USA) coupled with an automated Dionex Ulti-Mate 3000 UHPLC system controlled by Chromeleon 6.8 SR13 (Dionex Corporation, Part of Thermo Fisher Scientific) software.Full-scan HESI-MS and MS/MS acquisitions were performed in positive mode of the Q-Exactive Orbitrap mass spectrometer.The following parameters were used for the Orbitrap mass spectrometry techniques: auxiliary gas of 2; sheath gas of 40; capillary temperature of 300 °C; ion spray voltage of 3.2 kV; S-lens RF of 30 V; full-scan mode at a resolution of 70,000 m/z; mass range of 200-2000 m/z; top-20 data dependent MS/ MS acquisitions at a resolution of 35,000 m/z; and injection time of 35 min; automatic gain control target of 5e5; isolation window of 1 m/z; collision energy of 35 (arbitrary unit).The external calibration of instrument was performed to 1 ppm using ESI positive and negative calibration solutions (Thermo Scientific, Berkeley CA, USA).Mixtures of lipid standards were used to optimize tune parameters (Avanti Polar Lipids, Alabaster, AL, USA) in both positive and negative ion modes.Identification and semi-quantification of the classes of lipids and lipid molecular species present in the root and stem of both soybean cultivars (OX760-6 and Conrad) were performed using LipidSearch version 4.1 (Mitsui Knowledge Industry, Tokyo, Japan) and the parameters adopted for identification in LipidSearch were: target database of Q-Exactive; product tolerance of 5 ppm; precursor tolerance of 5 ppm; Quan m/z tolerance of ± 5 ppm; product ion threshold of 5%; m-score threshold of 2; Quan retention time range of ± 1 min; use of all isomer filter; ID quality filters A, B, and C; and [M + NH 4 ] + adduct ions for positive ion mode.Following identification, the observed lipid classes and lipid molecular species were merged and aligned according to the parameters established in our previous report [98].

Lipid biochemical network mapping
To better understand how soybean cultivars that are tolerant and susceptible to P. sojae modulate their membrane lipid mediators as part of the plant defense response strategy during infection and colonization, lipids that changed significantly between treatments were visualized within lipid structural similarity and implied activity networks.Lipid SMILES identifiers obtained from lipid map were used to calculate PubChem molecular fingerprints describing lipids' sub structures [99].Connections between lipids were defined based on Tanimoto similarity ≥ 0.8 between fingerprints.Significance of fold changes in lipid expression levels were mapped to network node attributes and displayed using Cytoscape [100,101].Node size was used to represent fold changes of means between treatments, and colors indicated the direction of change compared to control (orange = increased; blue = decreased; gray = inconclusive) in the lipid network map generated.Node shape was used to indicate lipid structural type (rounded square = membrane lipids; circle = neutral lipids).Lipids displaying significant differences between treatment groups (p ≤ 0.05) were denoted with black borders.

Statistical analysis
To determine the effects of pathogen infection on lipid composition of the root and stem of susceptible and tolerant cultivars, multivariate analyses including partial least square discriminant analysis (PLS-DA), and heat map were performed to group the treatments based on similarity.Analysis of variance (ANOVA) was next performed to determine whether the groups were significantly different between treatments using XLSTAT (2017 Premium edition, Addinsoft, Paris, France).Where significant differences were observed, the means were compared with Fisher's Least Significant Difference (LSD), α = 0.05.Figures were prepared with SigmaPlot 13.0 (Systat Software Inc., San Jose, CA).

Fig. 1 Fig. 2 Fig. 3
Fig. 1 Scanning electron microscopy (SEM) demonstrating morphology of soybean root segments.Tiny opening in the cortical cells indicated by red circle (a-c; 40-200 µm), intact inner cortical side of epidermis and cortex (d, e; 10, 40 µm), degradation and crack in the inner cortical side of epidermis and cortex (f, g; 40 µm), closing-up of degradation and crack in the inner cortical side of epidermis and cortex (h, i; 10 µm).Bar charts indicating the relative abundance of soybean root segments as mean ± SE (j; n > 5).Significant differences between morphology of soybean root segments are indicated using letters a-d on top of the bars following means separation using Fisher's LSD and ANOVA to assess significance of the model (α = 0.05)

Fig. 7
Fig. 6 Differences in root membrane lipids in susceptible (OX760-6) and tolerant (Conrad) soybean cultivars inoculated with P. sojae relative to control plants.a Model quality for partial least squares-discriminant analysis (PLS-DA); (b) Observation plot based upon differences in molecular species in root membrane lipids of OX760-6 and Conrad cultivars; (c) Heat map demonstrating clusters of root membrane lipid species in OX760-6and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated root membrane lipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of root membrane lipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are root membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root membrane lipid species in each of the bar chart (Fig.5d) beside the heat map; and (d) Bar charts describe the relative abundance of root membrane lipid species as a mean nmol% ± SE (n = 4).Significant differences between root membrane lipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are root membrane lipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root membrane lipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected)

Fig. 8
Fig. 8 Differences in root glycerolipid species in susceptible (OX760-6) and tolerant (Conrad) soybean cultivars inoculated with P. sojae relative to control plants.a Model quality for partial least squares-discriminant analysis (PLS-DA); (b) Observation plot based upon differences in molecular species in root glycerolipid species of OX760-6 and Conrad cultivars; (c) Heat map demonstrating clusters of root glycerolipid species in OX760-6and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated root glycerolipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of root glycerolipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are root glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root glycerolipid species in each of the bar chart (Fig.7d) beside the heat map; (d) Bar charts describe the relative abundance of root glycerolipid species as a mean nmol% ± SE (n = 4).Significant differences between root glycerolipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are root glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) root glycerolipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected)

Fig. 9
Fig. 9 Differences in stem glycerolipid species in susceptible (OX760-6) and tolerant (Conrad) soybean cultivars inoculated with P. sojae relative to control plants.a Model quality for partial least squares-discriminant analysis (PLS-DA); (b) Observation plot based upon differences in molecular species in stem glycerolipid species of OX760-6 and Conrad cultivars; (c) Heat map demonstrating clusters of stem glycerolipid species in OX760-6and Conrad cultivars treated or untreated with P. sojae.Each cultivar and treatment were grouped separately using ascendant hierarchical cluster analysis based upon Euclidian distance at interquartile range of 0.15.The left columns denote the cluster segregated stem glycerolipid species, while the above columns segregated soybean cultivars based upon similarities in abundance.The abundance of stem glycerolipid species is denoted using color: red for lower level, black for intermediate level, and green for higher level.Group 1 and 2 (G1 and G2) and subgroups (G1A, G1B, G2A and G2B) are stem glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem glycerolipid species in each of the bar chart (Fig.8d) beside the heat map; and (d) Bar charts describe the relative abundance of stem glycerolipid species as a mean nmol% ± SE (n = 4).Significant differences between stem glycerolipid species are indicate using letter a-d on top of the bars as described by Fisher's LSD multiple comparisons test using ANOVA (α = 0.05).The G1 and G2, and G1A, G1B, G2A and G2B are stem glycerolipid species that were accountable for the formation of clustered patterns in the heat map that were applied for the determination of significant differences between the soybean cultivars (OX760-6 and Conrad) stem glycerolipid species as illustrated in the bar charts.The susceptible cultivar, ORC (non-infected) & ORI (infected), and the tolerant cultivar CRC (non-infected) & CRI (infected)

Fig. 11
Fig. 11 Lipid structural similarity network displaying differences in stem membrane lipids and glycerolipids in susceptible and tolerant soybean cultivars inoculated with P. sojae relative to control plants.a Control susceptible soybean cultivar (OSC) versus inoculated (OSI); b control tolerant soybean cultivar (CSC) versus inoculated (CSI).The biochemical lipid network demonstrates fold differences in 21 stem membrane lipid molecular species and 28 glycerolipid molecular species following inoculation with P. sojae.Lipid SMILES identifiers were used to calculate PubChem molecular fingerprints and structural similarities.Mapped networks, displaying significance of fold differences in lipids were calculated for all comparisons.Network visualizations display lipids connected based on structural Tanimoto similarity ≥ 0.8 (edge width: 0.8 to 1.0).Node size displays fold differences of means between comparisons and color shows the direction of change compared to control (orange: increased; blue: decreased; gray: inconclusive).Node shape displays lipid structural type (rounded square: membrane lipids; circle: glycerolipids).Lipids displaying significant differences between treatment groups (p ≤ 0.05) are denoted with black borders

Table 1
Effect of Phytophthora sojae infection on the root lipidome of susceptible and tolerant soybean cultivarsValues in the table (nanomole% by weight composition) denote means ± standard errors for four biological replicates.Means in the same row with different superscripts are indicated as significantly different (*significant at alpha = 0.05) or not significantly different (ns) between the treatments, which consisted of susceptible control (OSC) and inoculated (OSI) stem tissue; and tolerant control (CSC) and inoculated (CSI) stem tissue from 10-day old seedlings, at a significance level of α < 0.05.The lipids detected were: PA Phosphatidic acid, PE Phosphatidyl-ethanolamine, PC Choline, PG Glycerol, PS Serine, PI Inositol, TG Triacylglycerol, DG Diacylglycerol,LPC Lysophosphatidylcholine, LPE Lysophosphatidylethanolamine, SM Sphingomyelin, MGDG Monogalactosyldiacylglycerol, SiE Beta sitosterol, StE Stigmasterol ester, HexCer Hexosyl ceramide, Cer Ceramide, CmE Campesterol ester, AcHexStE Acylated hexosyl stigmasterol ester, AcHexSiE Acylated hexosyl betasitosterol ester, and AcHexCmE Acylated hexosyl campesterol ester.Lipids that were not detected (ND) under the treatment conditions are indicated

Table 2
Effect of Phytophthora sojae infection on stem lipidome of susceptible and tolerant soybean cultivars Values in the table (nanomole% by weight composition) denote means ± standard errors for four biological replicates.Means in the same row with different superscripts are indicated as significantly different (*significant at alpha = 0.05) or not significantly different (ns) between the treatments, which consisted of susceptible control (OSC) and inoculated (OSI) stem tissue; and tolerant control (CSC) and inoculated (CSI) stem tissue from 10-day old seedlings, at a significance level of α < 0.05.The lipids detected were: PA Phosphatidic acid, PE Phosphatidyl-ethanolamine, PC Choline, PG Glycerol, PS Serine, PIinositol,TG triacylglycerol, DG Diacylglycerol, LPC Lysophosphatidylcholine, LPE Lysophosphatidylethanolamine, SM Sphingomyelin,MGDG Monogalactosyldiacylglycerol, SiE Beta sitosterol, StE Stigmasterol ester, HexCer Hexosyl ceramide, Cer Ceramide, CmE Campesterol ester, AcHexStE Acylated hexosyl stigmasterol ester, AcHexSiE Acylated hexosyl betasitosterol ester, and AcHexCmE Acylated hexosyl campesterol ester.Lipids that were not detected (ND) under the treatment conditions are indicated